EP2944311A1 - Combination of biologically active substances for treating hyperglycemic diseases - Google Patents

Abstract

The present invention relates to a synergistic composition comprising (a) phlorizin and (b) at least one more defined inhibitor of the enzyme lactase phlorizin hydrolase. In particular, the present invention relates to such a composition for the treatment and / or prophylaxis of a hyperglycemic disease, e.g. Obesity, diabetes or diabetes-related sequelae. The invention further relates to the use of such a composition for the preparation of a pharmaceutical composition, a dietetic food and / or a dietary supplement. The invention further relates to pharmaceutical compositions, dietetic foods and nutritional supplements comprising a composition according to the present invention.

Description

The present invention relates to a synergistic composition comprising (a) phlorizin and (b) at least one more defined inhibitor of the enzyme lactase phlorizin hydrolase. In particular, the present invention relates to such a composition for the treatment and / or prophylaxis of a hyperglycemic disease, e.g. Obesity, diabetes or diabetes-related sequelae. The invention further relates to the use of such a composition for the preparation of a pharmaceutical composition, a dietetic food and / or a dietary supplement. The invention further relates to pharmaceutical compositions, dietetic foods and dietary supplements comprising a composition according to the present invention.

FIELD OF THE INVENTION

According to the Federal Statistical Office, the number of diabetic deaths has increased by 29 percent since 1980. Diabetes mellitus is thus responsible for almost three percent of all deaths (1980: two percent) in Germany. At the same time, the number of overweight people in Germany is rising sharply. Every second German is already overweight, one in five is even obese. For these people, the risk of developing diabetes is dramatically increased.

Current projections assume that up to 7% of Germans ( Hauner H. Dtsch Med Wochenschr 2005; 130 Suppl 2: p.64-65 ), ie about 6 million people are being treated for diabetes. The number of new cases increases with age. In the age group over 60 years the proportion of diabetics is already 18-28%. In parallel, the age of onset of affected type 2 diabetics is steadily decreasing. At around 11%, type 2 diabetes is the fourth most common diagnosis of primary care physicians and, with around 8%, the fifth most common diagnosis of general practitioners ( Wittchen HU, (HYDRA) -study, Fortschr Med Orig 2003; 121 ). Current estimates from WHO estimate that the number of people with diabetes will double by 2025 ( World Health Organization, Diabetes mellitus, Fact sheet no. 138, 2002 ).

With food, a variety of fats, carbohydrates and proteins are taken up, which provide the human body with the required energy. Excessive uptake, and in particular too rapid intake of sugars, places a heavy burden on metabolism and is considered a major risk factor for diabetes, obesity and obesity. There is therefore a continuing need for new agents for the treatment of hyperglycemic diseases such as diabetes and obesity.

Numerous classes of compounds have been proposed for the treatment of diabetes mellitus, in particular type II diabetes mellitus. In various international applications, for example, substances have been proposed which inhibit the sodium-dependent glucose transporter 2 (SGLT-2) (see, eg WO 98/31697 . WO 02/083066 . WO 03/099836 and WO 01/31697 ). This transport protein resorbs glucose and sodium from the primary urine in the proximal tubule of the kidney. Specific SGLT-2 inhibitors are able to prevent the uptake of glucose into the renal tubules, resulting in the restoration of normal plasma glucose levels. Long-term treatment with SGLT-2 inhibitors of more than 6 months has been reported to increase insulin sensitivity, improve insulin response, and increase animal insulin sensitivity led to a delayed education of diabetes-related complications. The inhibition of the transporter SGLT-2 is based on an increased concentration-dependent excretion of glucose, which leads to a lowering of the blood sugar in the body. However, no hypoglycaemia is caused. The mechanism of lowering blood sugar is independent of any existing insulin resistance or lack of insulin production by the beta cells of the pancreas. In addition to SGLT-2, there is another sodium-dependent glucose transporter, SGLT-1, which has also been implicated in the treatment of diabetes. SGLT-1 is found in the small intestine as well as in the S3 segment of the proximal renal tubule. He is responsible for the active uptake of glucose. Compared to SGLT-2, SGLT-1 has altered substrate specificity for some sugars. Several drugs that inhibit SGLT-1 and / or SGLT-2 are currently in clinical trials.

The European patent application 12174534.3 describes the use of a composition containing an extract of a plant of the genus Brassica in combination with phlorizin or a phlorizin-containing extract. The composition is suggested for the treatment of hyperglycemic disorders.

DESCRIPTION OF THE INVENTION

In the context of the present invention, it has now been found that a beneficial, synergistic effect on the blood sugar level can be obtained if, in addition to an inhibition of the sodium-dependent glucose transporter SGLT1 by phlorizin, an inhibition of the enzyme lactase phlorizin hydrolase (LPH) by a In the present case described inhibitor takes place. A combined dose of phlorizin and one as defined herein LPH inhibitor leads to a significantly lower increase in blood glucose concentration after the intake of foods with a high proportion of carbohydrates, such as starch, and is therefore highly suitable for the treatment and / or prophylaxis of a metastatic hyperglycemic disease such as diabetes and / or overweight.

The enzyme LPH is well described in the art and is synthesized in the intestine of mammals. The enzyme is present as an integral membrane protein in the brush border membrane of the major cells of the villus epithelium of the small intestine of all mammals. It catalyzes in the intestinal lumen the cleavage of beta-glycosidic bonds in carbohydrates, e.g. Lactose. In addition to the hydrolysis of phlorizin, the LPH is also able to split the disaccharide lactose into the monosaccharides galactose and glucose. These two different activities of the enzyme take place at different reactive sites of LPH. In particular, according to the present invention, an inhibitor of LPH is capable of inhibiting the phlorizin-cleaving enzymatic activity of LPH, preferably human LPH. It is preferred that the inhibitor reduce this activity of the LPH by at least 5%, 10%, 15%, more preferably at least 20%, 25%, 30% or more preferably at least 40%, 50% 60%, 70% or 80% % reduced. The skilled worker knows of methods by which the activity of LPH and, correspondingly, its inhibition can be determined. Such methods are further described in the examples of the present application.

The LPH inhibitor (s) used in the present invention are flavonol glycosides which have a quercetin or kaempferol skeleton and are linked by a glycosidic bond to at least one sugar group. Consequently, it is in the Inventive proposed inhibitors to flavonol glycosides.

ist, in der Y ausgewählt ist aus der Gruppe bestehend aus H und OH; und wobei der Inhibitor mindestens eine glycosidisch an das Flavonol gebundene Zucker-Gruppe aufweist.In a first aspect, the invention relates to compositions containing at least the following components: (a) phlorizin and (b) at least one inhibitor of lactase-phlorizin hydrolase, wherein the inhibitor is a glycoside of a flavonol having the structure according to formula I.

where Y is selected from the group consisting of H and OH; and wherein the inhibitor has at least one glycosidically linked to the flavonol sugar group.

Flavonols are a subgroup of the chemical group of flavonoids. Flavonoids belong to the group of phytochemicals, and they represent an extensive class of polyphenolic compounds, which are common in foods of plant origin.

In the context of the invention, it has now been found that glycosides of a flavonol of the formula I are effective inhibitors of LPH. The formula I given above shows the basic structure of the flavonols kaempferol and quercetin. If Y is H, Formula I corresponds to Kaempferol. If Y is an OH group, formula I corresponds to quercetin. It is thus preferred according to the invention that the inhibitor of LPH is a glycoside of kaempferol or a glycoside of quercetin.

The substance of the formula I which acts as an inhibitor of the LPH has at least one glycosidic sugar group bound to the flavonol. Therefore, the LPH inhibitor is a glycoside of the formula I. Glycosides in the sense of the present invention are compounds which comprise a glycosidically bonded sugar residue and have the general structure R-O-Z, where Z is the sugar residue and R is the aglycone. Any type of sugar that can bind to formula I via a glycosylic bond can be used in the LPH inhibitors according to the invention. The glycosidically linked group may e.g. a mono- or a polysaccharide, e.g. a disaccharide or a trisaccharide.

It is preferred according to the invention that the glycosidically bound group is a monosaccharide. For example, the monosaccharide glycosidically bound to the flavonol of formula I may be a monosaccharide selected from the group consisting of glucose, fructose, rhamnose, glucopyranose, mannose, and galactose. The monosaccharide is preferably glucose. Thus, in a preferred embodiment, the present invention relates to a composition comprising (a) phlorizin and (b) at least one inhibitor of LPH, wherein the inhibitor is a glycoside of a flavonol of formula I and wherein Y is selected from the group consisting of H and OH; and wherein the inhibitor has at least one glycosidically linked to the flavonol glucose group. Accordingly, in this embodiment, the inhibitor of LPH is a glucoside. A glucoside is a compound comprising a glycosidically linked glucose residue and having the general structure RO-Glc, where Glc is a glucose residue and R is the aglycone.

In another preferred embodiment, the sugar group glycosidically linked to the compound of formula I is a polysaccharide. Suitable polysaccharides include e.g. Di-, tri-, tetra- and pentasaccharides. Suitable disaccharides are e.g. Cellobiose, gentiobiose, isomaltose, isomaltulose, lactose, lactulose, laminaribiose, maltose, maltulose, melibiose, neohesperidose, neotrehalose, nigerose, rutinose, sambubiose, sophorose, sucrose, and trehalose. Suitable trisaccharides are e.g. Fucosidolactose, Gentianose, Isokestose (1-Kestose), Kestose (6-Kestose), Maltotriose, Manninotriose, Melezitose, Neokestose, Panose, Raffinose, and Umbelliferose. In a preferred embodiment, the polysaccharide bound to the flavonol is rutinose.

The glycosidically bound sugar group is preferably bound to the flavonol beta-glycosidically. It is particularly preferred that the glycosidic bond of the sugar group via one of the OH groups shown in formula I in position 3 ', 4', 3, 5 or 7 takes place. The numbering of the positions in the flavonol follows the well-known chemical nomenclature, as indicated in the following formula II:

In a preferred embodiment, the glycosidically linked group of the inhibitor is in position 7 of the flavonol. In a further preferred embodiment is located the glycosidically bound group of the inhibitor is in position 3 of the flavonol. In yet another preferred embodiment, the inhibitor comprises only a single glycosidically linked sugar group located in position 3 of the flavonol. In yet another preferred embodiment, the inhibitor comprises only a single glycosidically linked sugar group located at position 7 of the flavonol. This means that in these embodiments, the inhibitor in addition to the glycosidically linked group in position 3 or 7 of the flavonol contains no further glycosidically bonded group.

According to the invention, the LPH inhibitor may also comprise a plurality of sugar groups which are each bonded via a glycosic bond to the flavonol according to formula I. The LPH inhibitor may e.g. 2, 3, 4, 5 or more such bound sugar groups. These glycosidically bound sugars are also preferably bound via one of the OH groups shown in formula I in position 3 ', 4', 3, 5 or 7.

The inhibitor of the LPH may in particular be one of the following molecules: quercetin-7-O-glucoside, kaempferol-7-O-glucoside, quercetin-3-O-rhamnoside, quercetin-3-O-glucopyranoside, kaempferol- 3-O-glucoside, kaempferol-3-O-rutinoside, quercetin-3-galactoside, quercetin-4'-glucoside, quercetin-3-O-rutinoside (quercetin-3-O-glucorhamnoside), kaempferol-3,7- di-O-α-L-rhamnoside, kaempferol-3- (2G-xylosylrutinoside), 3-glucosylquercetin, 3-glucosylkaempferol, dihydrokaempferol-3-rhamnoside, quercetin-3,4-O-glucoside, 4'-methylkaempferol, kaempferol 3-O-feruloyl diglucoside 7-O-glucoside, kaempferol 3-O-hydroxyferuloyl diglucoside, kaempferol 3-O-disinapoyl triglucoside 7-O-diglucoside, kaempferol 3-O-sinapoyl triglucoside , Kaempferol-3-O-sinapoyl-diglucoside, Kaempferol-3-O-disinapoyl-triglucoside-7-O-glucoside, Kaempferol-3-O-diglucoside-7-O-diglucoside, Kaempferol-3-O-triglucoside-7 -O-glucoside, Kaempferol-3-O-glucoside-7-O-glucoside, kaempferol-3-O-triglucoside, kaempferol-3-O-diglucoside, kaempferol-3-O-glucoside, and the like.

In a particularly preferred embodiment, the LPH inhibitor is quercetin-7-O-glucoside. In another embodiment, the LPH inhibitor is kaempferol-7-O-glucoside.

In addition, the LPH inhibitors of the invention may also be SGLT1 inhibitors, thus exhibiting a dual action. Examples of such dual inhibitors are e.g. Quercetin-7-O-glucoside and kaempferol-7-O-glucoside.

The glycosidic linked to the flavonol sugar group may have further modifications. Thus, in one embodiment, the sugar group is acylated, i. the sugar group comprises one or more acyl groups. Preferred acylated inhibitors of lactase phlorizin hydrolase are e.g. selected from the group consisting of kaempferol-3-O-hydroxyfexuloyltetraglucoside, kaempferol-3-O-hydroxyferuloyl-diglucoside-7-O-hydroxyferuloyl diglucoside, camphorol-3-O-hydroxyferuloyl-diglucoside-7-O-glucoside, kaempferol- 3-O-sinapoyl-diglucoside-7-O-diglucoside and kaempferol-3-O-sinapoyl-diglucoside-7-O-glucoside. In a particularly preferred embodiment, the inhibitor is kaempferol-3-O-sinapoyl diglucoside-7-O-diglucoside.

It is preferred according to the invention that the glycosidic sugar group bound to the flavonol is an acylated polysaccharide.

In addition, a sugar group attached to the flavonol may be a cinnamic acid derivative, ie the sugar group comprises one or more cinnamic acid derived groups such as a hydroxycinnamic acid group such as a sinapic acid group (3,5-dimethoxy-4-hydroxycinnamic acid ), α-cyano-4-hydroxy-cinnamic acid group (HCCA), ferulic acid group (4-hydroxy-3-methoxycinnamic acid) and caffeic acid group. In this embodiment, the sugar group is present for example as sinapoyl glycoside or as feruloyl glycoside.

The compositions according to the invention comprise, in addition to one or more inhibitors of LPH, the flavonoid phlorizin. Phlorizin inhibits the transporter SGLT 1 in vitro ( Kottra et al. (2007), J Pharmacol Exp Ther, 322 (2): 829-35 ). It has also been known for a long time that phlorizin acts on the renal glucose excretion when administered intravenously. However, the effect is weak when phlorizin is taken orally. This is presumably partly due to enzymatic degradation of phlorizin in the intestine, which is catalysed, for example, by the enzyme LPH ( Birkenmeier & Alpers (1974), Biochim Biophys Acta., 350 (1): 100-12 ). After ingestion of the composition of the invention, enzymatic degradation of intestinal phlorizin is inhibited by the LPH inhibitor (s) defined above, whereby the phlorizin exhibits its glucose uptake inhibitory properties (SGLT 1).

The compositions of the invention contain an amount of phlorizin which is effective to inhibit the glucose transporter SGLT-1 after oral administration and / or to slow sugar uptake from the intestine. In a preferred embodiment, the activity of the intestinal SGLT-1 transporter is increased by the compositions by at least about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50 %, 60%, 70%, 80%, 90% or more reduced compared to the untreated condition. In a further preferred embodiment, the composition according to the invention gives a sugar uptake from the intestine compared to the untreated state after administration by at least about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40 %, 50%, 60%, 70%, 80%, 90% or more.

The person skilled in the art is easily able to determine the amount of phlorizin and LPH inhibitor required for the respective (therapeutic) effect to be achieved by simple series of experiments. Here, the skilled person will consider various factors, such as the nature and amount of the inhibitor or inhibitors of LPH present in the composition, the nature and amount of the components additionally present in the compositions, the age and weight of the person to whom the administration of the composition takes place, etc.

The amount of phlorizin in the compositions of the invention is preferably greater than 1 ppm (parts per million) or 0.0001% (w / w). It is preferred that the amount of phlorizin in the composition is greater than 10 ppm, greater than 100 ppm, greater than 1000 ppm, greater than 10 ⁴ ppm, greater than 5 * 10 ⁴ ppm or greater than 10 ⁵ ppm. In other words, the composition according to the invention contains more than 0.0001% (w / w) more than 0.001%, preferably more than 0.01%, more than 0.1%, more than 1%, more than 2%, more than 3 %, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 11%, more than 12%, more than 13 %, more than 14%, or more than 15% phlorizin. It is also preferred that the composition according to the invention contains more than 20% (w / w), more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more comprises 55%, or more than 60% phlorizin. In a particularly preferred embodiment, the compositions of the invention contain between 1-95% (w / w), such as 5-80%, 10-70%, 15-60%, 20-50%, or 30-40% phlorizin, or such as 5-95%, 10-95%, 15-95%, 20-95%, or 30-95% phlorizin.

The composition according to the invention is preferably formulated such that the respective administration unit (eg a capsule, a tablet or a defined amount of a liquid) contains an amount of 0.01 to 10 g of phlorizin, eg more than 0.05 g, more than 0.1 g, more than 0.2 g, more than 0.3 g, more than 0.4 g, more than 0.5 g, more than 0.75 g, more than 1 g, more than 2 g, more than 3 g, more than 4 g or more than 5 g.

The compositions of the invention further contain an amount of LPH inhibitor effective to inhibit lactase phlorizin hydrolase after oral administration. In a preferred embodiment, the intestinal LPH activity is increased by the compositions by at least about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%. , 70%, 80%, 90% or more decreased from the untreated condition.

The amount of LPH inhibitor in the compositions of the invention is also preferably greater than 1 ppm (parts per million) or 0.0001% (w / w). It is preferred that the amount of LPH inhibitor in the composition is greater than 10 ppm, greater than 100 ppm, greater than 1000 ppm, greater than 10 ⁴ ppm, greater than 5 * 10 ⁴ ppm, or greater than 10 ⁵ ppm. In other words, the composition according to the invention contains more than 0.0001% (w / w), more than 0.001%, preferably more than 0.01%, more than 0.1%, more than 1%, more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 11%, more than 12%, more than 13%, more than 14%, or more than 15% LPH inhibitor. It is also preferred that the composition according to the invention contains more than 20% (w / w), more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more as 55%, or more than 60% of the one or more LPH inhibitors defined herein. In a particularly preferred embodiment, the compositions of the invention contain between 1-95% (w / w), such as 5-80%, 10-70%, 15-60%, 20-50% or 30-40% LPH inhibitor, or such as 5-95%, 10-95%, 15-95%, 20-95%, or 30-95% LPH inhibitor.

In one embodiment, the LPH inhibitor present in the above amounts in the composition of the invention is kaempferol-7-O-glucoside. In another embodiment, the LPH inhibitor present in the composition of the invention in the above amounts is quercetin-7-O-glucoside.

The composition of the invention is preferably formulated such that the particular administration unit (e.g., a capsule, powder, tablet or defined amount of liquid) contains an amount of from 0.01 to 10 g of LPH inhibitor, e.g. more than 0.05 g, more than 0.1 g, more than 0.2 g, more than 0.3 g, more than 0.4 g, more than 0.5 g, more than 0.75 g, more than 1 g, more than 2 g, more than 3 g, more than 4 g or more than 5 g.

It is particularly preferred that the composition according to the invention is formulated for oral administration and contains from 0.01 to 10 g LPH inhibitor, preferably from 1-3 g LPH inhibitor, and from 0.01 to 10 g phlorizin, preferably from 1 -3 g phlorizin.

Phlorizin and LPH inhibitor (s) can be used in any mixing ratio. However, the amounts of phlorizin and LPH inhibitor in the compositions according to the invention are preferably combined by the person skilled in the art in such a way that an optimal effect is achieved, ie both LPH and SGLT1 are efficiently inhibited. In one embodiment, the composition of the invention contains phlorizin and LPH inhibitor (s) in the ratio of approximately 20: 1, 10: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1: 1, 1: 2 , 1: 3, 1: 4, 1: 5, 1:10 or 1:20. A mixing ratio of phlorizin and the LPH inhibitor (s) of 1: 1 is particularly preferred.

In a particularly preferred embodiment, the LPH inhibitor is not added to the composition according to the invention in the form of a plant extract. If the LPH inhibitor originated from a plant extract, the inhibitor was purified from the extract prior to its use in the compositions of the present invention. Alternatively, the LPH inhibitor may also be a compound obtained by chemical synthesis. Methods for the chemical synthesis of the above-mentioned inhibitory flavonol compounds are known to the person skilled in the art and have been described in the literature, see eg Hirpara et al. (2009, Anticancer Agents Med Chem, 9 (2): 138-61 ).

Furthermore, it is preferred that the LPH inhibitors and phlorizin as defined above total more than 70% (w / w), preferably more than 80%, 85%, 90%, 95% or 99% of the flavonoid present in the composition according to the invention Make up glycosides. This means that less than 30% (w / w), preferably less than 20%, 15%, 10%, 5% or 1% flavonoid glycosides are present in the composition according to the invention, which are not those defined above LPH inhibitors or phlorizin. In a particularly preferred embodiment, the composition according to the invention contains less than 1% (w / w) or no flavonoid glycosides, which are not the above-defined LPH inhibitors or phlorizin.

It is particularly preferred according to the invention that both the phlorizin used in the compositions according to the invention and the LPH inhibitor are synthetically produced. In a further particularly preferred embodiment, neither the phlorizin nor the LPH inhibitor are present in the form of a plant extract.

In another embodiment, the phlorizin is added to the LPH inhibitor (s) in the form of a phlorizin-containing plant extract. The phlorizin-containing herbal extract can be obtained, for example, from at least one plant of the family Rosaceae . Preferably, the plant of the family Rosaceae belongs to a genus selected from the group consisting of Malus, Pyrus or Prunus. The phlorizin-containing herbal extract may also be obtained from a plant of the family Verbenaceae . Preferably, the plant of the family Verbena ceae is one of the genus Lippia. It is known that especially plants of these families contain phlorizin. The extract of the plant of the family Rosaceae or Verbenaceae can be obtained, for example, from the bark, the fruits or the leaves of the plant.

Plants of the genera Malus, Pyrus and Prunus contain relatively high amounts of phlorizin in the bark, in the fruits and in the leaves. In particular, the fruits of the genus Malus (ie apples) have particularly high levels of phlorizin. It is therefore particularly preferred that the phlorizin is present in the composition according to the invention in the form of an extract from a plant of the genus Malus, preferably in the form of an extract from apples or the bark. The genus Malus is a plant genus in the family Rosaceae. It is particularly preferred that the extracts are produced from a plant of the genus Malus starting from plants of the species Malus domestica . The cultivated apple (Malus domestica Borkh, Pyrus malus, L.) is an economically very important type of fruit, which includes numerous varieties. In principle, all varieties of the culture apple can be used to prepare the phlorizin-containing extracts. It is particularly preferred that the extracts starting from one of the varieties Red Delicious, Golden Delicious, Braeburn, Cox Orange, Finkenwerder Prince, Prince Blücher, Mark Cox or Red Chief. In addition to plants of the species Malus domestica , however, other species of the genus Malus can be used.

It is preferred that the at least one extract from the plant of the family Rosaceae and / or the at least one extract from the family of Verbenaceae at least 1%, preferably at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or more phlorizin contains (w / w).

In a further preferred embodiment, the compositions comprise one or more inhibitors according to the invention of the LPH and at least one extract of at least one plant of the family Rosaceae and / or of at least one plant of the family Verbenaceae. In one embodiment, the compositions contain one or more, for example two, three, four or more, of the inhibitors of LPH according to the invention and / or more than one, eg two, three, four or more, extracts from a plant of the genus Malus. Particularly preferred are compositions containing quercetin-7-O-glucoside and / or kaempferol-7-O-glucoside and at least one extract of a plant of the genus Malus . As used herein, the term "extract from a plant" refers to an extract derived from one or more plants of the same species and is not to be understood as limited to a single plant.

As used herein, the term "extract" refers to a composition of matter obtained by digesting cells of a plant and recovering the cell juice. The extracts proposed by the invention can be prepared by a variety of methods known in the art.

In one embodiment of the invention, material of a plant is first provided. As starting material for the inventive Methods are suitable for plants at all stages of development, from seeds to mature plants. Furthermore, all plant parts, such as the fruits, stems, leaves, bark or roots of the plant are suitable. The starting material may be a mixture of different plant parts, or may only include certain parts of plants. The vegetable material is usually crushed first. The crushing can be achieved by simply breaking up the respective plant parts with conventional cutters. In principle, all plant parts can be used as a starting point for extract formation. In the production of extracts of plants of the genus Malus , the fruits and the bark have proven to be particularly suitable. In a further preferred embodiment, the extracts are produced from a plant of the genus Malus from the fruits, ie the apples, or from the bark of the respective malus plant. Prior to comminution, the vegetable material may be blanched at temperatures between 45 ° C and 100 ° C to remove bacterial contamination and improve the quality of the digestion. Furthermore, blanching reduces the activity of enzymes such as hydrolases, lipases and oxidases and thus increases the stability and quality of the plant material.

In a next step, the plant material is disrupted, ie the cellular structures of the material are destroyed, so that the content of the cells is released. Digestion may be achieved by conventional plant cell disruption techniques, such as by repeated freezing and thawing, or by suitable equipment such as homogenizers, high pressure homogenizers, or ultrasonic homogenizers. Colloid mills or French presses can also be used for digestion.

In addition, the cell disruption can also be achieved enzymatically. For this purpose, the plant material is mixed with suitable enzymes, which lead to a destruction of the structural components of the cells. It has been found that incubation of the plant material with pectinase, collagenase, cellulase and / or hemicellulases in this phase of the method is particularly suitable for effectively disrupting the cells. The incubation time can be between 30 minutes and 24 hours, preferably between 1 hour and 6 hours, more preferably between 90 minutes and 4 hours, e.g. 2 hours. The temperature may be in the range of 20 ° C and 60 ° C, preferably between 25 ° C and 45 ° C, e.g. at about 37 ° C, and it should preferably be in the range of the temperature optimum of the particular enzyme used. A suitable pH of the reaction mixture may be determined using suitable buffers such as e.g. Phosphate, carbonate, sodium bicarbonate buffer and / or suitable acids, e.g. Citric acid, lactic acid or ascorbic acid can be adjusted. A pH between 4 and 6, e.g. pH 5, is particularly suitable for digestion.

After digestion of the cells, the resulting cell sap can be used directly to inhibit the sodium-dependent glucose transporter 1 (SGLT-1). However, it is preferred to subject the vegetable juice obtained after the digestion of the cells to further purification steps.

For example, the material obtained from the cell disruption may be subjected to drying or freeze-drying with subsequent extraction. Here, for example, first a spray-drying can be used. The dried or freeze-dried plant material may then be extracted with an aqueous or organic solvent. The extraction can be carried out with a conventional extractant, for example with ethanol, ethyl acetate or with other organic solvents. Also, an extraction with gases, such as with nitrogen is possible. When using a liquid solvent, the incubation time is 0.5 to 24 hours, preferably 2 to 8 hours. When using nitrogen, the extraction is carried out at temperatures between 4 ° C and 37 ° C in a period of 0.25 to 3 hours, preferably in a period of 20 and 60 minutes.

The compositions proposed by the invention can be prepared by a variety of methods known in the art. The LPH inhibitors and phlorizin of the present invention can be prepared by methods known in the art or purchased directly from various manufacturers and mixed together in the desired amounts (e.g., Carl Roth GmbH & Co. KG, Karlsruhe, Germany).

In a preferred embodiment, the phlorizin is concentrated in the phlorizin-containing extract. Processes for concentrating these substances are well known to the person skilled in the art and are described, for example, in US Pat Will et al. (2006, LWT - Food Science and Technology, 40 (8): 1344-1351 ). Concentration of phlorizin can be achieved, for example, by using column chromatography techniques. For example, different Adsorberäulen can be used for this purpose. The chromatographic columns are loaded with the starting extract, freed of unwanted constituents by rinsing with suitable solutions and then eluted from the column in concentrated form. For example, after loading adsorbent columns with unconcentrated extract, by rinsing with demineralized water, hydrophilic substances (salts, amino acids, peptides, sugars, etc.) are depleted. Phlorizin is then enriched in the extract eluted with 95% ethanol. The phlorizin extracts obtainable according to the invention can be produced both in dry and in liquid form.

The compositions according to the invention are proposed according to the invention for the treatment and / or the prophylaxis of a hyperglycemic disease. The compositions are characterized by a synergistic effect of their constituents, i. the interaction of the individual components goes beyond a purely additive effect.

Diseases that can be treated with the compositions proposed by the invention include all diseases caused by hyperglycemia, i. are characterized by an elevated blood sugar level that exceeds the physiologically normal value of 140 mg / dl (7.8 mmol / l). Such diseases include, in particular, obesity, diabetes or diabetes-related sequelae (such as retinopathy, neurophathy, nephropathy, and impaired wound healing). The diabetes to be treated by the compositions of the present invention may be Type I or Type II diabetes. Preferably, it is Type II diabetes. Further, the compositions of the invention are generally suggested for the treatment and / or prophylaxis of such diseases and conditions in which it is advantageous to slow and / or reduce sugar absorption from the intestine.

The compositions proposed according to the invention for the treatment or prophylaxis can also be used advantageously with other active substances, eg together with other antidiabetic agents described in the prior art, such as biguanides, sulphonylureas, glycosidase inhibitors, preferably inhibitors of carbohydrate-cleaving enzymes of the digestive tract (eg. 4,6-Dideoxy-4 - [[1S- (1S, 4R, 5S, 6S) -4,5,6-trihydroxy-3- (hydroxymethyl) -2-cyclohexen-1-yl] -amino] -alpha D-glucopyranosyl- (1-> 4) -O-alpha-D-glucopyranosyl- (1-> 4) -D-glucopyranose (please refer DE 23 47 782 ), Thiazolidinediones, dipeptidyl peptidase IV (DP4) inhibitors, meglitinides, glucagon-like peptide-1 (GLP-1), PTP1B inhibitors, glycogen phosphorylase inhibitors, glucose-6-phosphatase inhibitors, and SGLT1 and SGLT2 inhibitors described in the following property rights: WO 02/080936 . WO 02/080935 . JP 2000080041 . EP 0 850 948 (Propiophenonglckoside); WO 02/044192 . WO 02/028872 . WO 03/011880 and WO 01/068660 (2-Glucopyranoslyoxybenzylbenzole); WO 02/068440 . WO 02/068439 . WO 02/36602 . WO 01/016147 . WO 02/053573 . WO 03/020737 . WO 03/090783 . WO 04/014932 . WO 04/019958 and WO 04/018491 (Glucopyranosyloxypyrazole); WO 01/074835 and WO 01/074834 (O-Glycosidbenzamide); WO 04/007517 (Glucopyranosyloxythiophene); WO 03/099836 . WO 01/027128 and US 2002/0137903 (C-aryl glycosides); DE 102 58 008 (Fluorine-glycoside derivatives); DE 10 2004 028 241.2 (Fluoro-glycoside derivatives of pyrazoles).

The compositions of the invention containing at least phlorizin as well as one or more LPH inhibitors will in the present case be formulated for common or sequential administration. Therefore, the present invention also relates to the use of phlorizin and one or more LPH inhibitors of the invention for the treatment and / or prophylaxis of a hyperglycemic disease, wherein the components are formulated for separate administration.

In one aspect, the present invention relates to the use of a composition as described above for the preparation of a pharmaceutical composition, a dietetic food or a nutritional supplement.

In a still further aspect, the present invention thus also relates to a pharmaceutical composition, a dietetic A food or dietary supplement comprising a composition as described above comprising at least one LPH inhibitor and phlorizin of the invention. The pharmaceutical composition, dietetic food or dietary supplement are suitable for use in a method for the treatment and / or prophylaxis of a hyperglycemic disease, preferably selected from the group consisting of obesity, diabetes or diabetes-related sequelae, and in particular for the treatment and / or or prophylaxis of type II diabetes.

A pharmaceutical composition comprising the composition of the invention may be formulated for oral, parenteral or topical administration. Such a pharmaceutical composition may be prepared by methods well known in the art. Such processes as well as suitable auxiliaries and carriers are described, for example, in US Pat. Remington: The Science and Practice of Pharmacy ", Lippincott Williams & Wilkins, 21st Edition (2005 ). The pharmaceutical compositions may be in the form of, for example, granules, powders, tablets, capsules, syrups, suppositories, injections, emulsions, suspensions or solutions. The compositions may be formulated for various modes of administration, for example, for oral, parenteral, topical, buccal, sublingual, transmucosal, rectal, subcutaneous intrathecal, intravenous, intramuscular, intraperitoneal, nasal, intraocular or intraventricular administration. The formulation as oral, parenteral or topical composition is particularly preferred. In a particularly preferred embodiment, the composition is formulated for oral administration. The pharmaceutical compositions may also be formulated as sustained-release agents.

For oral, buccal and sublingual administration, solid formulations such as, for example, powders, suspensions, granules, tablets, pills, capsules and gelcaps are generally used. These can be prepared, for example, by mixing the active ingredients (the phlorizin and the one or more inhibitors of the invention lactase phlorizin hydrolase) with at least one additive or with at least one excipient. Such auxiliaries and carriers are described, for example, in US Pat. Remington: The Science and Practice of Pharmacy ", Lippincott Williams & Wilkins, 21st Edition (2005 ). As additives or auxiliaries, for example, microcrystalline cellulose, methylcellulose, hydroxypropylmethylcellulose, casein, albumin, mannitol, dextran, sucrose, lactose, sorbitol, starch, agar, alginates, pectins, collagen, glycerides or gelatin can be used. Further, compositions for oral administration may include antioxidants (eg, ascorbic acid, tocopherol, or cysteine), lubricants (eg, magnesium stearate), preservatives (eg, paraben or sorbic acid), flavor enhancers, disintegrants, binders, thickeners, dyes, and the like.

Liquid formulations of the compositions according to the invention which are suitable for oral administration can be present for example as emulsion, syrup, suspension or solutions. These formulations may be prepared using a sterile liquid carrier (eg, oil, water, alcohol, or combinations thereof) in the form of liquid suspensions or solutions. For oral or parenteral administration, pharmaceutically suitable surfactants, suspending agents, oils or emulsifiers may be added. Suitable oils for use in liquid dosage forms include, for example, olive oil, sesame oil, peanut oil, rapeseed oil and corn oil. Suitable alcohols include ethanol, isopropyl alcohol, hexadecyl alcohol, glycerine and propylene glycol. suspensions may further comprise fatty acid esters such as ethyl oleate, isopropyl myristate, fatty acid glycerides and acetylated fatty acid glycerides. Furthermore, suspensions are often mixed with substances such as mineral oil or petrolatum.

The present invention further relates to a dietetic food or a dietary supplement comprising a composition as defined above, ie a composition containing phlorizin and one or more inhibitors of the invention of LPH. The dietary food or dietary supplement is particularly useful for the treatment and / or prophylaxis of obesity as defined above, diabetes (especially Type II diabetes) or diabetes-related sequelae. The phlorizin may be derived from at least one phlorizin-containing, vegetable extract. The extract is preferably from a plant of the genus Malus, in particular from a plant of the species Malus domesticus. It is particularly preferred that at least one extract from a plant of the genus Malus has been obtained from the apples or the bark of a Malus plant.

EXAMPLES

The following examples describe the effect of the compositions according to the invention:

Example 1: In vitro inhibition of LPH by quercetin and kaempferol derivatives

Various quercetin and kaempferol derivatives have been tested in vitro as inhibitors of lactase phlorizin hydrolase (LPH). The test was carried out after Amiri et al. (2012, J Inherit Metab Dis, 35 (6): 949-54 ). Here, tissue of the human small intestine (0.6 mg / ml) expressing the LPH was used. The substrate used was phlorizin dihydrate. The released glucose was determined by means of a standard assay ( Chu & Cheung, 1978, Clin Biochem, 11 (4): 187-9 ).

Results:

The quercetin and kaempferol derivatives tested showed a high inhibitory activity on the LPH and were able to inhibit LPH in a concentration-dependent manner (Table 1). Quercetin-7-glucoside and kaempferol-7-O-glucoside showed a particularly intense inhibition of LPH by about 70% at a concentration of 50 μg / ml. <b> Table 1: Inhibition of various glycosidases by quercetin and kaempferol derivatives; nb: not determined. </ b> No. Name inhibitor Synonyms Inhibition of LPH (10 μg / ml inhibitor) Inhibition of LPH (50 μg / ml inhibitor) 1 quercitrin Quercetin-3-O-rhamnoside nb 12.8% (100 μg / ml) 2 Quercetin-3-glucopyranoside Quercetin-3-0-glucopyranoside nb 13.7% (100 μg / ml) 3 Quercetin-7-glucoside 3,3 ', 4', 5,7-pentahydroxyflavone-7-glucoside 44.0% 69.4% 4 Kaempferol-3-0-glucoside astragalin 3.3% 45% 5 Kaempferol-7-0-glucoside Populnin 53.0% 70.5% 6 Kaempferol-3-0-rutinosid nicotiflorin (+ 7.2%) 6.6% 7 hyperosid Quercetin-3-galactoside 18.4% 26.2% 8th spiraeoside Quercetin-4'-glucoside 31.3% 44.1% 9 RUTIN Quercetin-3- O- rutinoside Quercetin-3- O- glucorhamnoside 34.7% 36.0% 10 KAEMPFERIT-RIN Kaempferol-3,7-di-O-α-L-rhamnoside; 3,4 ', 5,7-tetrahydroxyflavone-3,7-dirhamnosid 19.8% 18.4% 11 QUERCETIN-3-GLUCOSIDE 5.00 3-Glucosylquercetin; 3,3 ', 4', 5,7-pentahydroxyflavone-3-glucoside 21.6% 38.6% 12 Kaempferol-3-glucoside 3-Glucosylkaempferol; 3,4 ', 5,7-tetrahydroxyflavone-3-glucoside 18.5% 11.7%

Example 2: Inhibitory effect of a combination of phlorizin and LPH inhibitor on LPH / SGLT1

The effect of a combination of phlorizin and an LPH inhibitor on the uptake of glucose by the SGLT 1 transporter was tested on an ex vivo model that simulates the situation in the human gut. The ex vivo test model uses human small intestinal tissue that expresses lactase phlorizin hydrolase (LPH) and thus is able to cleave phlorizin and other glycosides. Subsequently, the preprocessed in this way experimental approaches were tested in an in vitro assay for their inhibitory effect on the SGLT1 transporter.

Human small intestinal tissue expressing lactase phlorizin hydrolase (LPH) was as in Amiri & Naim, described and prepared (J Inherit Metab Dis (2012) 35: 949-954 ). In summary, so-called "brush border membranes" were prepared from the small intestine and incubated in a suitable buffer with phlorizin and / or the respective LPH inhibitor for 90 minutes.

Subsequently, the small intestinal membranes were separated and the activity of SGLT1 in the supernatants after a through Kottra & Daniel described electrophysiological procedures (J Pharmacol Exp Ther. 2007; 322 (2): 829-35 ). Xenopus oocytes expressing human SGLT1 were used. The transport of glucose by SGLT1 can be measured because the co-transport of glucose and sodium by SGLT1 causes flow through the oocyte membrane. This is determined electrophysiologically by means of a "two-electrode voltage clamp" method. Reduction of the activity of SGLT1 in this model also leads to a reduction of the current across the cell membrane.

Results:

It has been shown that phlorizin and the tested LPH inhibitors synergistically reduce the activity of SGLT. Table 2 shows the inhibition by the compounds tested with the respective inhibition values in percent of the original activity. Surprisingly, the combination of phlorizin and the tested LPH inhibitors kaempferol-7-O-glucoside and quercetin-7-glucoside, respectively, was able to reduce the activity of the SGLT significantly more than the individual substances in total (see, eg, approach A). While phlorizin, quercetin-7-glucoside, and kaempferol-7-O-glucoside alone each reduced glucose uptake by 32%, 2%, and 2.5%, respectively, the combinations of phlorizin and quercetin-7-glucoside and phlorizin and Kaempferol 7-O-glucoside able to reduce glucose uptake by 48% and 52%, respectively.

The same effect was also observed when an apple extract (batch B) containing 15% phlorizin was used instead of the pure phlorizin. This extract inhibited SGLT activity by 33%. Quercetin-7-glucoside and kaempferol-7-glucoside alone reduced the activity of SGLT1 by 2.5% and 2.0%, respectively, in this approach. In contrast, the combination of the apple extract with quercetin-7-glucoside or kaempferol-7-glucoside achieved an inhibition of 49% and 54%, respectively. <b> Table 2: </ b> Inhibition of SGLT1-mediated glucose uptake in an ex <i> vivo </ i> model Approach A Glucose uptake, inhibition Phlorizin 0.3 μg / ml 32% Quercetin-7-glucoside 10 μg / ml 2% Quercetin-7-glucoside 10 μg / ml & phlorizin 0.3 μg / ml 48% Kaempferol 7-O-glucoside 10 μg / ml 2.5% Kaempferol 7-O-glucoside 10 μg / ml & phlorizin 0.3 μg / ml 52% Approach B Apple extract (with 15% phlorizin) 33% 2 mg / ml Quercetin-7-glucoside 10μg / ml 2.5% Apple extract (with 15% phlorizin) 2 mg / ml & quercetin-7-glucoside 10 μg / ml 49% Kaempferol 7-O-glucoside 10 μg / ml 2.0% Apple extract (with 15% phlorizin) 2 mg / ml & kaempferol 7-O-glucoside 10 μg / ml 54%

Claims (13)

Composition comprising: (a) phlorizin; and (b) one or more inhibitors of lactase phlorizin hydrolase, wherein the inhibitor is a glycoside of a flavonol having the structure according to formula I.

where Y is selected from the group consisting of H and OH; and
wherein the inhibitor has at least one glycosidically bound to the flavonol sugar group. The composition of claim 1, wherein the glycosidically linked group of the inhibitor is in position 3 or position 7 of the flavonol, wherein the glycosidically linked group is preferably linked beta-glycosidically. A composition according to claims 1-2, wherein the sugar group is a glucose group. A composition according to claim 2-3, wherein in addition to the glycosidically linked group in position 3 or position 7 of the flavonol no further glycosidically bonded group is present. The composition of claim 4, wherein the lactase phlorizin hydrolase inhibitor is selected from the group consisting of quercetin-7-O-glucoside and kaempferol-7-O-glucoside. A composition according to any one of claims 1-5, wherein the composition comprises from 0.01 to 10 g of phlorizin. A composition according to any one of claims 1-6, wherein the composition comprises from 0.01 to 10 g of the inhibitor of lactase phlorizin hydrolase. A composition according to any one of claims 1-7 for use in a method for the treatment and / or prophylaxis of a hyperglycemic disease, wherein the disease is preferably selected from the group consisting of obesity, diabetes or diabetes-related sequelae. Use of the composition according to any one of claims 1-7 for the preparation of a pharmaceutical composition, a dietetic food or a nutritional supplement. A pharmaceutical composition, dietetic food or dietary supplement comprising the composition according to any one of claims 1-7. A pharmaceutical composition, dietetic food or nutritional supplement comprising the composition according to any one of claims 1-7 for use in a method for the treatment and / or prophylaxis of a hyperglycemic disease, preferably selected from the group consisting of obesity, diabetes or diabetes-related sequelae. A composition for use according to claim 8, or a pharmaceutical composition, dietetic food or dietary supplement for use according to claim 11, wherein the diabetes is Type II. A pharmaceutical composition according to claim 10, or a pharmaceutical composition for use according to claim 11 or 12, characterized in that it is formulated for oral, parenteral or topical administration.